Antileishmanial Activity of Lavandula angustifolia and Rosmarinus Officinalis Essential Oils and Nano-emulsions on Leishmania major (MRHO/IR/75/ER).

Background
The aim of present study was to evaluate antileishmanial effects of Lavandula angustifolia (L. angustifolia) and Rosmarinus officinalis (R. officinalis) medicinal plants essential oils and nano-emulsions on Leishmania major (L. major).


Methods
The present study was performed in Leishmaniasis Reference Lab at Mazandaran University of Medical Sciences, Iran during 2016-2017. The IC50 values were calculated in both the promastigote and amastigote stages in J774 macrophage in comparison with meglumine antimoniate (MA) as positive control. In addition, cytotoxicity effects of essential oils and nano-emulsions prepared from both plants against macrophages were evaluated.


Results
Both essential oil and nano-emulsion of Lavander and Rosemary showed anti-leishmania activity on promastigote with IC50=0.11 μl/mL, IC50=0.26 μl/mL, and IC50=0.08 μl/mL respectively. Moreover, during amastigote assay, Lavander and Rosemary essential oils and nano-emulsion were effective at least in concentration of 0.12 μl/mL and 0.06 μl/mL (P=0.0001) respectively, on mean infected macrophages (MIR) and amastigotes in macrophages (P=0.0001). Additionally, cytotoxicity assay against macrophage revealed no toxicity on the host cells at IC50 concentrations.


Conclusion
The nano-emulsions of both plants were more effective than essential oil in both MIR and amastigote. However, in comparison with MA, the Lavander essential oil is more effective in reducing MIR. Rosemary nano-emulsion reduced MIR significantly more than MA in concentration of 0.25 μl/mL (P<0.001). Further investigations are recommended to evaluate the effect of these medicinal plants in murine models.


Introduction
eishmaniases are neglected vectorborne parasitic diseases caused by an obligatory intracellular parasite of Leishmania spp. The infection is transmitted by bite of female sand fly during taking blood meals (1). The disease is endemic in 98 countries on five continents. More than 90% of cutaneous leishmaniasis (CL) forms appear in Afghanistan, Saudi Arabia, Algeria, Brazil, Iran, Iraq, Syria, and Sudan. In addition, the most cases of visceral form reporting from India, Sudan, and Brazil (1-3). Over the last decade, the incidence of CL has increased in more than half of the provinces (20 out of 32) in Iran (4).
The efficacy of current regimes including pentavalent antimony as first-line drugs has decreased and drug resistance has increased in many parts of the world. Second line drugs such as amphotericin B and pentamidine are very toxic (5).
The urgent need for substitute treatments has led to a program for screening natural products in leishmaniasis. Actually, the WHO recommended the use of traditional medicine in communities with poor health services (6). Some of herbal plants were used in traditional medicine for long times and today we know that they have anti-leishmanial effects as well and could be considered candidates for new drugs (7)(8)(9).
Rosemary is an herbal plant, commonly used for food flavoring and useful for the treatment of several diseases as an anti-inflammatory agent (10). It is useful in prevention or treatment of respiratory problems, peptic ulcers, tension headache, renal colic, heart disease and spasmogenic disorders (11).
Anti-parasitic activity of Lavandula angustifolia and L. xintermedia essential oils against three human protozoal pathogens Giardia duodenalis, Trichomonas vaginalis and the fish pathogen Hexamita inflate were evaluated (12). The aim of present study was to investigate anti-leishmanial effects of Lavandula angustifolia and Rosmarinus officinalis essential oils and nanoemulsions on L. major.

Preparation of nano-emulsion
The present study was performed in Leishmaniasis Reference Lab at Mazandaran University of Medical Sciences (MAZUMS), Iran during 2016-2017. The oil-in-water (o/w) nano-emulsions of Lavander and Rosemary essential oils were prepared using essential oil (1% w/w) and Span 60 (0.5% w/w) as oil phase, and mixture of Tween 80 (1% w/w) in deionized water as aqueous phase. The nanoemulsions were formulated as described previously (13). The physicochemical characterization was defined in terms of mean particle size, poly dispersity index and zeta potential using Zeta sizer Nano ZS (Malvern Instruments, UK). The results are the means of three determinations.

Gas Chromatography and Mass Spectrometry
Gas chromatographic analysis was carried out on a Perkin-Elmer 8500 gas chromatograph with FID detector and a DB-5 capillary column (30 m  0.25 mm; film thickness 0.25 m).
Gas Chromatography-Mass Spectrometry (GC-MS) was performed on Hewlett Packard 6890 series, using a DB-5 capillary column (30 m  0.25 mm, film thickness 0.25 m) programmed as follows: 60 C for 5 min and then up to 220 C at 4 C/min. The carrier gas was helium at a flow rate of 2 mL/min. The carrier gas was helium at a flow rate of 2 mL/min; split ratio, 1: 40; ionization energy, 70 eV; scan time, 1 sec; acquisition mass range, m/z 40-400.
Identification of Components: The components of the oil were identified by their retention time, retention indices relative to C 9 -C 28 n-alkanes, computer matching L with the WILEY275.L library and as well as by comparison of their mass spectra with those of authentic samples or with data already available in the literature (14,15).

Promastigote assay
The in vitro evaluation of the antileishmanial activity of essential oils of the Lavandula angustifolia and Rosmarinus officinal and nano-emulsions of these plants, on parasites in the promastigote stage, were assessed using 96-well microplate. For the determination of the 50% inhibition concentration, each well was filled with 100 μL of the parasites suspension (1×10 6 parasites/mL). Consequently, 10 μL of serial dilutions (from 1 to 0.0625 µl/mL) of selected plant essential oils and nano-emulsions were added to the same wells of microplate then the plate was incubated at 26 °C for 72 h. Wells without any component used as negative control and meglumine antimoniate (MA) (Glucantime®, Rhône-Poulenc, France) in well used as positive one.
In brief, 10 ml of MTT (5 mg/mL) was added to each well and plates were further incubated for 4 h. The enzyme reaction was then stopped by addition of 100 µL of 50% isopropanol-10% sodium dodecyl sulfate. The plates were incubated for an additional 30 min under agitation at room temperature. Three replicates for each exposure concentration were examined. Absorbance values at 570 nm MTT (corrected at 630 nm) were measured using a microplate spectrophotometer (Bio-RAD Benchmark Plus). As a control, the activity of a drug alone in reagent was determined, and no substantial interaction was found. Then, The IC 50 values of MA, Lavandre and Rosemary for promastigotes were calculated using the following formula:

Amastigote assay
Macrophage line J774 A.1 (ATCC number TIB-67) was obtained from National Cell Bank of Iran (Pasteur Institute, Tehran, Iran). Macrophages were kept in RPMI-1640 medium. Cells were diluted in medium then following which viability test was performed by adding 90 μl of trypan blue solution (0.2%) in saline containing 0.01% sodium aside to 10 μL of cell suspension (10 6 cells / mL). After 2 min, cells were counted under light microscope, where viability calculated as follows: %Viability = (% of live cells /all counted cells) × 100 Briefly, 200 μL of the cells (10 6 cells /mL) was added into 8chanel-chamber slides (SPL.Korea) and incubated at 37 °C with 5% CO 2 for 2 h for cell adhesion. Promastigotes (10 7 /mL) were added to macrophages and incubated at 37 °C with 5% CO 2 for 24 h. Then essential oils and nano-emulsions in several concentrations (10 μL) in medium added to the slides and incubated at 37 °C for 72 h. In addition, MA as a reference drug was added.
Dried slides were fixed with ethanol, stained with Wright-Giemsa and studied under light microscope.
Macrophages containing amastigotes with no drugs and macrophages alone were considered as positive and negative controls, respectively. Drug activity was evaluated by counting the number of amastigotes in the macrophages by examining 100 macrophages and the number of macrophages was infected in 100 macrophages (MIR) (17).

Cytotoxicity assay
In vitro toxicity against J774.A.1 macrophages was assessed with cells plated in 96well plates at 2 × 10 5 cells ⁄ well. After cell adherence, the medium was removed and replaced by the media containing IC 50 concentration of each compound. The plates were incubated for 24 h at 37 °C in an incubator with 5% CO 2 . Control cells were incubated with culture medium plus DMSO. Cell viability was determined using MTT colorimetric assay.

Data analysis
Indicator absorbance values at 72 h time points for MTT were analyzed using calculated using CalcuSyn version 2 software (Biosoft, UK). In addition, the SPSS software was used to analyze the data. ANOVA test, multiple comparison test, and t-test were used and P<0.05 was considered as a significant difference.
The in vitro anti-leishmanial activities of the nano-emulsions of Lavander and Rosemary as well as essential oils were investigated against standard strain of L. major. For Lavander essential oil and nano-emulsion, the effective concentration with IC 50 =0.11 μl/mL achieved. For Rosemary essential oil, the effective concentration with IC 50 =0.26 μl/mL and its nano-emulsion IC 50 =0.08 μl/mL were achieved. The IC 50 value of MA was 197 mg/ mL, is significantly higher than those of Lavander and Rosemary nano-emulsions and essential oils (P< 0.0001).

Amastigote activity
The effect of essential oil and nanoemulsion of both Lavander and Rosemary on amastigote stage of parasite was evaluated by the mean infection rate of macrophages (MIR) and by the mean number of amastigotes per macrophage. Comparison of the MIR showed that different concentrations of Lavander and Rosemary essential oils significantly inhibited infection of macrophages by at least 0.12 μl/ Ml (P<0.0001) ( Table 1). For nano-emulsion the effective concentration which significantly inhibited infection of macrophages were at least 0.06 μl/mL when compared with positive control (P<0.001). Moreover, comparing different concentrations of Lavander and Rosemary essential oils on the mean number of amastigotes in each macrophage was 0.12 μl/ mL as for nano-emulsion the mean number of amastigotes in each macrophage decreased at the concentration of 0.06 μl/ mL when compared with positive control (P<0.001) ( Table  2). All results were compared with MA, which was the drug of choice in our study. The difference between Lavander essential oil and MA in reducing mean infection rate of macrophages (MIR) was significant in doses 1 μl/ mL (P=0.001) and 05 μl/ mL (P=0.044).  In these concentrations, MIR was declined to 19.33% and 24.33% respectively which was lower than MA with 30.33%. However, in terms of the difference between Rosemary essential oil and MA in reducing MIR, same result achieved in reduced MIR. Rosemary essential oil reduced MIR to 13.33 % and 23.33, which was lower than MA with 30.33%. (P=0.0001). Rosemary essential oil in concentration of 1 μl/mL significantly decreased MIR more than Lavander (P=0.0001). Lavander and Rosemary nano-emulsions significantly reduced MIR and the number of amastigotes in macrophages in at least 0.06 μl/mL in compare with control (P=0.001). The difference between several concentrations of Rosemary nano-emulsion in concentration of 0.25 μl/mL in reducing amastigotes in macrophages and MA was significant (P=0.04). Moreover, Rosemary essential oil in concentration of 1μl/mL decreased amastigotes in macrophages significantly more than MA (P=0.04). Rosemary nano-emulsion reduced amastigotes in macrophages in concentrations of 0.25.0.125 and 0.0625 μl/mL significantly more than Lavander nano-emulsion (P=0.39) ( Table 2).

Discussion
Pentavalent antimonials as first-line drugs had been widely used for the treatment of CL all around the world for decades, but treatment failure occurs in endemic regions (18). Alternative drugs such as miltefosine, amphotericin B, and some azoles are used as second-line drugs for the treatment of disease (19). All of these drugs have serious side effects and are expensive and unavailable in some endemic areas and there are drugresistant Leishmania strains (20).
There is an urgent need for new and safe components and drugs, therefore medical plants can be an alternative (20,21). The use of plants for treating disease is as old as human history and frequently prescribed during the decades. The extracted components are used all around the world to treat health disorders and disease such as infectious forms (22). Plant extracts are widely used in Iran. Currently, about 820 herbal drugs are produced in Iran. Efficacy of some herbal plants like garlic, shallots, wormwood, yarrow, walnuts, thyme, henna plant, mimosa, aloe, wood betony and figwort have been shown on Iranian strain of L. major (MRHO/IR/75/ER) by In vitro and In vivo assessments (20,23). However, the efficacy of some herbal plants has not been evaluated clinically and scientifically, but the interest for the use of plants, as safer and cheaper therapy exists (21).
L. major has been associated with zoonotic cutaneous leishmaniasis (ZCL) and it has high incidence rate in Iran (18). In present study, we evaluated in vitro anti-leishmanial effect of essential oils and nano-emulsions of Lavander and Rosemary. Rosemary extract has shown antitumor property as it can prevent cell proliferation. The researchers isolated carnosol and carnosic acid from Rosemary leaves which had anticancer property (24). In addition, Rosemary has antibacterial and anti-fungal effect and its anti-oxidant and anti-proliferative activity have been evaluated previously (24,25). Lavander oils as antibacterial agents display good antibacterial activity against a range of bacteria including Streptococcus, pyogenes, Enterobacter aerogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Citrobacter freundii, Proteus vulgaris, Escherichia coli, VRE, Shigella sonnei and Propionibacterium acnes (12). Our study revealed the sufficient effect of these components on L. major promastigote and amastigote stages. This is the first time that essential oils and nano-emulsions of Lavander and Rosemary evaluated on L. major in Iran. We found different sensitivity of this parasite to the component assayed. Several concentrations of Lavander and Rosemary essential oils had sufficient effect on promastigote and amastigote stages of L. major in at least IC 50 = 0.11 μl/mL and IC 50 =0.26 μl/mL respectively. In addition, nano-emulsions of Lavander and Rosemary affected promastigote and amastigote stages of L. major in at least IC 50 =0.11 μl/mL and IC 50 =0.08 μl/mL respectively. Both essential oil and nano-emulsion of Lavander on at least IC 50 =0.11 μl/mL affected the promastigote stage of L. major although Rosemary nanoemulsion had lower IC 50 and it had more inhibitory effect on L. major. The IC 50 of Rosemary essential oil is 4 times less than Lavander. In addition, IC 50 of Lavander essential oil is 0.32 times more than Rosemary nano-emulsion. Results were compared with MA with IC 50 =197mg/mL and revealed that essential oils and nano-emulsions of both Lavander and Rosemary are more effective than MA.
In amastigote stage of parasite, Lavander and Rosemary essential oils were effective at least in concentration of 0.12 µl/mL and significantly reduced MIR (P=0.0001)( Fig. 1. A,B). In addition, Lavander and Rosemary nano-emulsion reduced MIR by at least 0.06 µl/mL which was significantly lower than control (P=0.0001). Obviously, the effective concentration of nano-emulsion was lower than essential oil in both components to reduce MIR ( Fig. 1. C,D). Nano-emulsion of both Lavander and Rosemary has the same effect on decreasing the rate of infected macrophages but there is a little difference in concentration of 0.25µl/mL and Rosemary nano-emulsion was more effective than Lavander in this concentration (13.33% versus 37.33%). Lavander essential oil significantly decreased the number of amastigotes in macrophages compared with control at least in 0.12 µl/mL, the difference between several concentrations and MA was not significant (P>0.05) ( Fig.  2.A)( Table 2). Even in concentrations of 1µl/mL and 0.5 µl/mL of Lavander essential oil, MA decreased the number of amastigotes in macrophages significantly more. Results were achieved for Rosemary essential oil revealed in concentration 1µl/mL, the number of amastigotes in macrophages were significantly lower than MA (P=0.04) (Fig. 2. B) ( Table 2). Lavander and Rosemary nano-emulsions significantly decreased the number of amastigotes at least in 0.12 µl/mL (P=0.002) and 0.06 µl/mL (P=0.0001) in comparison with control ( Fig. 2.C and D). Rosemary nanoemulsion is more effective than Lavander nanoemulsion in decreasing the number of amastigotes. After all, nano-emulsions were effective in lower concentrations than essential oils in both MIR and amastigotes in macrophages considered efficient components, but when we compared them with drug of choice (MA), Rosemary essential oil and nanoemulsion were more effective than Lavander in reducing MIR in concentration 0.25 µl/mL (29.6% versus 32%)( P=0.49) (Fig. 1. C). Lavander and Rosemary essential oils were compared and in concentrations 1 µl/ml, 0.5 µl/ml, 0.25 µl/ml Rosemary essential oil decreased the number of amastigotes in macrophages significantly more than Lavander (P=0.04).
Nanoemulsions of Lavander and Rosemary were compared and statistically significant dif-ference observed as in concentrations 0.12 µl/mL and 0.06 µl/mL, the number of amastigotes in macrophages with Rosemary nano-emulsion was less than Lavander (P=0.04) (Fig. 2 C and D). There are limited studies carried out on Lavander and Rosemary extracts.
In a study carried out on atopic dermatitis in induced mice, the researchers evaluated the effect of Lavander and thyme essential oils on oxidative stress and immunity in disease. Their results showed the efficient effect of Lavander essential oil and the mixture with thyme in controlling disease symptoms (26). Another study with Rosemary extracts on young rats revealed that the extract had no effect on enhancing general immunity but may be effective in some conditions of stress like anti-oxidant or protein deficiency (27).
Lavander has antiparasitic effect as in primary investigation with two Lavander including L. angustifolia and L. xintermedia essential oils against three protozoal pathogens Giardia duodenalis, and Trichomonas vaginalis and the fish pathogen Hexamita inflata, the efficacy of Lavander was evaluated. In addition, L. angustifolia essential oil has activity against mite, grain weevils, aphids, and clothes moth. Research on the use of essential oils and other plant extracts on protozoan pathogens are limited to Plasmodium spp and Leishmania spp (12,28).
According to our preliminary evaluation, essential oils and nano-emulsions of both Lavander and Rosemary decreased the number of infected macrophages and the number of amastigotes in macrophages compared with control, and in cases described earlier more than MA. In addition, Rosemary nano-emulsion was more effective than Lavander. Low concentration of nano-emulsions and essential oils compared with MA and amazing results showed the good efficacy of both Rosemary and Lavander nano-emulsions and essential oils on L.major. These formulations could be used with MA in combination therapy to reduce the time of healing.

Conclusion
The nano-emulsions of both L. angustifolia and R. officinalis medicinal plants were more effective than essential oil in both MIR and amastigote. As a whole, clinical assessment of the anti-leishmanial activity of these plants on murine model is recommended.